THE JOURNAL OF BIOLOGICAL CHEMISTRY © 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Vol. 279, No. 17, Issue of April 23, pp. 17674 –17680, 2004 Printed in U.S.A. Glucosylceramidase Mass and Subcellular Localization Are Modulated by Cholesterol in Niemann-Pick Disease Type C* Received for publication, December 10, 2003, and in revised form, January 30, 2004 Published, JBC Papers in Press, February 2, 2004, DOI 10.1074/jbc.M313517200 Rosa Salvioli‡, Susanna Scarpa§, Fiorella Ciaffoni‡, Massimo Tatti‡, Carlo Ramoni¶, Marie T. Vanier储, and Anna Maria Vaccaro‡** From the Departments of ‡Hematology, Oncology and Molecular Medicine, and ¶Cell Biology and Neuroscience, Istituto Superiore Sanita’, 00161 Roma, Italy, §Department of Experimental Medicine and Pathology, University of Rome ‘La Sapienza’, 00161 Roma, Italy, and 储INSERM Unit 189, Lyon-Sud Medical School, Oullins and Fondation Gillet-Mérieux, Lyon-Sud Hospital, 69495 Pierre-Bénite, France Niemann-Pick disease type C (NPC)1 is an autosomal-recessive neurovisceral lipid storage disorder (1). Most cases of NPC are caused by mutations in the NPC1 gene (2) encoding a protein which possesses a sterol-sensing domain (3). The putative function of NPC1 protein is to facilitate the recycling of lipids from late endosomes/lysosomes to other cellular membranes (4 – 6). High levels of unesterified lipoprotein-derived cholesterol (Chol) accumulate in NPC1-deficient cells. Although alterations of Chol metabolism play a key role in the pathogenesis of NPC, there is also a more general dysfunction of the intracellular metabolism of lipids such as sphingolipids * This work was partly supported by the Association “Vaincre les Maladies Lysosomales.” The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ** To whom correspondence should be addressed: Istituto Superiore di Sanita’, Viale Regina Elena 299, 00161 Roma, Italy. Tel.: 39-0649902416; Fax: 39-06-49387149; E-mail: avaccaro@iss.it. 1 The abbreviations used are: NPC, Niemann-Pick disease type C; Chol, unesterified lipoprotein-derived cholesterol; SL, sphingolipids; GC, glucosylceramide; GM2, GalNAc␤4(Neu5Ac␣3)Gal␤4Glc-ceramide; GCase, glucosylceramidase; DMEM, Dulbecco’s modified Eagle’s medium; LPDS, lipoprotein-deficient bovine serum; FBS, fetal bovine serum; LAMP1, lysosome-associated membrane protein type 1; LBPA, lysobisphosphatidic acid. (SL) (7–10). Spleen and liver of NPC patients accumulate not only Chol, but also glucosylceramide (GC), lactosylceramide, and sphingomyelin. Normal concentrations of Chol, but pathological levels of GC, lactosylceramide, GM2-ganglioside and asialo-GM2 in brain are typical findings (1). These observations indicate that the NPC1 protein may function in Chol and SL homeostasis. In normal cells, the SL are degraded in late endosomes/ lysosomes by specific hydrolases. Some of these enzymes need the assistance of activator proteins such as saposins to exert their function (11–13). Saposins are a group of four similar small glycoproteins, Sap A, B, C, and D, each of them stimulating the enzymatic degradation of specific SL. In fact, Sap B is required for the degradation of sulfatides by arylsulfatase A, and Sap C is required for the degradation of GC by glucosylceramidase (GCase) (14 –16). The physiological role of saposins has been unequivocally demonstrated by the observation that SL storage diseases can be caused either by the deficiency of a specific hydrolase or of an individual saposin. For instance, Gaucher disease, a genetic disorder characterized by an extensive GC accumulation within the lysosomes of cells of monocyte/macrophage origin, can be caused by a deficit of either GCase or Sap C (16). In the Sap C-deficient cases of Gaucher disease, normal levels of GCase are unable to degrade GC. The role of Sap C in the enzymatic GC degradation has been examined in detail. In the past, we have provided compelling evidence that Sap C, at low pH values mimicking the acidic lysosomal environment, tightly binds to and destabilizes anionic phospholipid-containing membranes (17). Upon affecting the physical organization of these membranes, Sap C promotes the association of GCase with the lipid surface, thus favoring the contact between the enzyme and its lipid substrate, GC (18, 19). Anionic phospholipids play a key role in the Sap C-promoted interaction of GCase with membranes; changes in the level and organization of these lipids can affect the topology and activity of GCase (18, 19). Markedly increased amounts of GC have been found not only in Gaucher disease, but also in visceral tissues and in brains from NPC patients (8, 20). Because SLs such as GC are believed to be centrally involved in the pathogenesis of NPC disease (21), the mechanism of their accumulation and the properties of the hydrolases involved in the SL degradation have been extensively investigated. For instance, it has been found that the activities of GCase and sphingomyelinase are markedly reduced in NPC fibroblasts (22). Chol-mediated regulation of sphingomyelinase activity has been investigated (23, 24), whereas informations on the regulation mechanism of GCase in NPC cells are not available. It is important to fill this gap, because the accumulation of GC is very pronounced in 17674 This paper is available on line at http://www.jbc.org Downloaded from http://www.jbc.org/ by guest on June 7, 2020 Niemann-Pick disease type C (NPC) is characterized by the accumulation of cholesterol and sphingolipids in the late endosomal/lysosomal compartment. The mechanism by which the concentration of sphingolipids such as glucosylceramide is increased in this disease is poorly understood. We have found that, in NPC fibroblasts, the cholesterol storage affects the stability of glucosylceramidase (GCase), decreasing its mass and activity; a reduction of cholesterol raises the level of GCase to nearly normal values. GCase is activated and stabilized by saposin C (Sap C) and anionic phospholipids. Here we show by immunofluorescence microscopy that in normal fibroblasts, GCase, Sap C, and lysobisphosphatidic acid (LBPA), the most abundant anionic phospholipid in the endolysosomal system, reside in the same intracellular vesicular structures. In contrast, the colocalization of GCase, Sap C, and LBPA is markedly impaired in NPC fibroblasts but can be reestablished by cholesterol depletion. These data show for the first time that the level of cholesterol modulates the interaction of GCase with its protein and lipid activators, namely Sap C and LBPA, regulating the GCase activity and stability. Modulation of Glucosylceramidase in NPC Cells several NPC tissues. The aim of our present work was to investigate the factors that might influence the GCase activity and stability in NPC cells. The possibility that the function of Sap C, a required cofactor for the enzymatic degradation of GC, might be altered in these cells was also taken into consideration and investigated. EXPERIMENTAL PROCEDURES values of total 35S-labeled cellular proteins were utilized for each experimental point. After the addition of 0.1% BSA, the cell lysates were incubated with rabbit preimmune serum overnight at 4 °C, and nonspecific complexes were precipitated with protein A-Sepharose CL-4B. The clarified supernatants were then incubated either with anti-GCase or anti-Sap C antiserum. Cross-reacting material was precipitated with protein A-Sepharose CL-4B. The immunocomplexes were washed four times with PBS containing 1% BSA, 1% Triton X-100, 1% SDS, 0.4% sodium deoxycholate, and then with only PBS. The washed precipitates were separated by SDS-PAGE. Labeled proteins were detected by fluorography. Fluorescence Microscopy—For fluorescence microscopy, the cells were grown on Labteck chamber slides (Nunc, Naperville, IL) and fixed with 4% paraformaldehyde in PBS for 30 min. Cells were then rinsed with PBS, permeabilized with 0.05% saponin for 7 min, and incubated with 3% bovine serum albumin for 2 h. For intracellular free unesterified Chol staining, fixed cells were incubated with filipin solution (0.05% in PBS) for 30 min. The cells were observed with a UV 330 –380 filter. For double immunostaining, the cells were incubated for 1 h with a specific rabbit polyclonal primary antibody (anti-Sap C or polyclonal anti-GCase), rinsed twice with PBS, and incubated for 1 h with the secondary anti-rabbit antibody conjugated with Alexa Fluor 594 (Molecular Probes, Eugene, OR). The cells were then rinsed twice with PBS, incubated for 1 h with a specific mouse monoclonal primary antibody (anti-GCase (8E4), anti-LAMP1, or anti-LBPA), rinsed twice with PBS, and incubated with the secondary anti-mouse antibody conjugated with Alexa Fluor 488 (Molecular Probes, Eugene, OR). Finally, the cells were mounted with ProLong antifade reagent (Molecular Probes) and observed with an Olympus BX52 fluorescence microscope equipped with appropriate filters. The images were acquired using the IAS 2000 software. When specified, the fluorescence was viewed by confocal laser-scanning microscopy using a Leica TCS 4D apparatus equipped with an argon-krypton laser, double-dichroic splitters (488/568 nm), 520-nm barrier filter for Alexa Fluor-488 (green), and 590-nm barrier filter for Alexa Fluor-594 (red) observations. Image acquisition and processing were conducted by using SCANware, Multicolor Analysis (Leica Lasertechnik, GmbH, Heidelberg, Germany), and Adobe Photoshop software programs. Signals from different fluorescent probes were taken in parallel, and colocalization was detected in yellow. Primary antibodies were used at the following dilutions: anti-Sap C (1:300), monoclonal anti-GCase (1:300), polyclonal anti-GCase (1:100), anti-LAMP1 (1:200), and anti-LBPA (1:80). RESULTS GCase Activity and Mass Are Reduced in NPC Fibroblasts— GCase activity has been reported to be markedly diminished in NPC cells (22). As shown in Fig. 1, the GCase activity was ⬃400 nmol/h/mg of protein in normal fibroblasts, whereas it was reduced to 75–100 nmol/h/mg protein (about 20% of the normal value) in cell lines from two NPC patients who lacked the NPC1 protein (NPC1a and NPC1b). Thus, much less functional GCase is present in NPC1 fibroblasts as a consequence of either a reduction or inactivation of the enzyme protein. To examine the first possibility, the GCase mass was analyzed by Western blotting using a monoclonal anti-GCase antibody. The intensity of the enzyme bands in both the NPC1 cell lines was ⬃80% weaker than in normal fibroblasts (Fig. 1, inset), indicating that much less protein was present. Thus, the difference in activity between the control and the mutated cells correlates well with differences in the enzyme mass. GCase Activity and Mass Are Modulated by Chol Accumulation—To address the possibility that the decreased amount of GCase is related to the accumulation of endolysosomal free Chol, the NPC1 cells were grown in LPDS medium. It is known that NPC cells no longer accumulate Chol when cultured for more than 2 days in lipoprotein-free medium (1). Accordingly, the free Chol level was dramatically reduced upon removal of low density lipoproteins, as indicated by the cytochemical filipin-staining of the NPC1 cells (data not shown). The GCase activity increased 3– 4 times after 7 days of subculture with LPDS (Fig. 2A), and a parallel increase of the GCase protein Downloaded from http://www.jbc.org/ by guest on June 7, 2020 Materials—CompleteTM (protease inhibitor mixture) was obtained from Roche Applied Science. Dulbecco’s modified Eagle’s medium (DMEM) was obtained from Euroclone Ltd, UK. [35S]methionine (Tran35S-Label™, 1175 Ci/mmol) and methionine/cysteine-deficient DMEM were obtained from ICN Biomedicals, Inc., Costa Mesa, CA. Lipoprotein-deficient bovine serum (LPDS) was obtained from Cocalico Biologicals, Inc. Filipin and protein A-Sepharose CL-4B were obtained from Sigma. Kodak X-Omat Blue films were from PerkinElmer Life Sciences. Prolong anti-fade kit was obtained from Molecular Probes (Eugene, OR). SDS-PAGE reagents were from Bio-Rad. ECL Western blotting reagents were from Amersham Bioscience, Buckinghamshire, UK. Cell Cultures—Two human fibroblast lines with previously described severe NPC1 mutations were used (25). The NPC1a cell line (81057) was homozygous for a Q775P mutation located in the sterol-sensing domain and shown to produce no detectable NPC1 protein by Western blot analysis. The NPC1b cell line (90089, affected sib of reported 87024) was homozygous for a V282fs mutation. Normal and NPC1 fibroblasts were cultured in DMEM supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, 100 units/ml of penicillin, and 100 ␮g/ml streptomycin. For specific experiments, NPC1 cells were first grown in DMEM supplemented with 10% FBS and then subcultured in fresh medium containing 10% LPDS for the indicated periods of time. GCase Assay—To measure the GCase activity, lipid substrate GC, purified from Gaucher spleens, was utilized (26). GC was labeled with tritium in the glucose moiety (27). The assay mixture contained in a final volume of 0.1 ml: 0.1/0.2 M citrate/phosphate buffer, pH 5.6, 10 ␮g of cell homogenate, 20 ␮g of GC supplemented with the 3H-labeled compound to a specific activity of 3000 dpm/nmol, 0.25% taurocholate, and 0.05% oleic acid. The assay mixtures were incubated for 1 h at 37 °C. The incubation was terminated by the addition of 0.4 ml of chloroform/methanol (2:1) and 50 ␮l of a 0.1% glucose solution. After shaking and centrifugation at 4000 rpm, the enzymatically released [3H]glucose present in the aqueous phase was estimated by radioactivity measurements. Antibodies—Mouse monoclonal (8E4) and rabbit polyclonal antiGCase antibodies were kindly provided by Dr. H. Aerts, E. C. Slater Institute for Biochemical Research, University of Amsterdam, The Netherlands. Rabbit anti-human Sap C antibody was prepared in our laboratory (17). Mouse monoclonal anti-human lysosome-associated membrane protein type 1 (LAMP1) antibody, developed by Dr. J. T. August, was obtained from the Developmental Studies Hybridoma Bank maintained by the University of Iowa (Iowa City, IA). Mouse monoclonal anti-lysobisphosphatidic acid (LBPA) antibody (6C4) was a generous gift of Dr. J. Gruenberg, Department of Biochemistry, University of Geneva, Switzerland. The anti-actin monoclonal antibody was obtained from Sigma. Western Blotting—SDS-PAGE was performed with 10% acrylamide gels (28). After electrophoresis, the proteins were electroblotted to polyvinylidene difluoride membranes (Bio-Rad), and GCase was detected with anti-GCase monoclonal antibody 8E4 using an ECL Western blotting kit, according to the manufacturer’s instructions (Amersham Bioscience, Buckinghamshire, UK). Metabolic Labeling and Immunoprecipitation of GCase or Sap C— Skin fibroblast cultures were grown until they almost reached confluency. Prior to being labeled, the cells were washed twice with ice-cold PBS supplemented with 1 mM MgCl2 and 0.1 mM CaCl2 and starved for 2 h in methionine and cysteine-free medium containing 4% dialyzed FBS. This medium was replaced with the labeling medium (DMEM lacking methionine and cysteine and supplemented with [35S]methionine, 150 ␮Ci/ml, and 4% dialyzed FBS). After a 1-h incubation, the cells were washed three times with DMEM and non-radioactive chase medium was added (DMEM containing 4% FBS). The cells were chased for the indicated periods and then harvested and disrupted in lysis buffer (0.5% Triton X-100 and a protease inhibitor mixture tablet/50 ml in 50 mM phosphate buffer, pH 6.5). The suspensions were subjected to brief sonication and centrifuged at 20,000 ⫻ g for 30 min. For removing DNA and histones, the supernatants were incubated with 0.03% protamine sulfate for 45 min at 4 °C and centrifuged as above. Constant 17675 17676 Modulation of Glucosylceramidase in NPC Cells was observed (Fig. 2B). When normal fibroblasts cells are grown in lipoprotein-free medium for 7 days, we observed a ⬃20% increase of the GCase activity (from about 400 to 450 – 480 nmol/h/mg), whereas in NPC1 cells, the increase of activity was ⬃250% (from about 100 to 300 –350 nmol/h/mg). Thus, the level of free Chol in the endolysosomal system is able to modulate the level of GCase protein. Maturation of GCase in NPC1 Cells—To investigate at which step of maturation the amount of GCase decreased in the NPC1 cells, the biosynthesis and processing of the enzyme was examined by pulse-chase experiments (Fig. 3). According to previous findings (29, 30), a GCase precursor form (about 62 kDa) in normal fibroblasts was observed after a pulse of 1 h. A band at higher molecular mass (about 65 kDa) appeared after a 24-h chase. The fibroblast chased for 72 h contained an additional 58-kDa band of mature GCase. A similar pattern was observed in NPC1 fibroblasts. The densitometric quantitation of the intensity of the bands revealed that the amount of the 62-kDa precursor formed during a 1-h pulse was essentially the same in both control and NPC1 cells. In contrast, after a 72-h chase, much less GCase was detected in NPC1 than in control fibroblasts. These results indicate that the stability of the mature forms of GCase is markedly decreased in NPC1 cells. Maturation of Sap C in NPC1 Cells—Sap C, a small glycoprotein (about 10 kDa) derived from a large molecular mass precursor, prosaposin (65–70 kDa) (31), is the specific activating and stabilizing factor of GCase (11). A possible cause of the GCase instability in NPC1 cells might be a reduced amount of Sap C. To test this hypothesis, the biosynthesis and maturation of Sap C have been examined. As shown in Fig. 4, the amount of the prosaposin 65- to 70-kDa forms detected after pulselabeling for 1 h and the amount of Sap C generated after 72 h of chase were nearly the same in normal and NPC1 fibroblasts. Thus, the instability of GCase in NPC1 cells simply cannot be ascribed to a lack of Sap C. Nevertheless, it must be noted that during a chase of 5 h, about 50% of prosaposin was converted to the mature saposin in normal fibroblasts, although only 5–15% was cleaved in NPC1 cells. This finding, consistent upon repetition, indicates that the prosaposin processing was retarded in the mutated cells. DISCUSSION In addition to an impairment in Chol trafficking, the NPC cells are characterized by an extensive endolysosomal accumulation of SL. Previous studies have shown that the activity of sphingomyelinase and GCase, which are responsible of the degradation of two SLs present at high concentrations in NPC tissues, namely sphingomyelin and GC respectively, are markedly reduced (22). Our present results show that the reduction of GCase activity is paralleled by a decrease of the enzyme mass, and that both activity and mass can revert to almost normal levels when the lipoprotein fraction is removed from the culture medium of NPC1 fibroblasts, namely when free Chol within the late endosomal/lysosomal compartment disappears. Thus, it is evident that Chol can modulate the level of GCase in NPC1 fibroblasts. Downloaded from http://www.jbc.org/ by guest on June 7, 2020 FIG. 1. GCase activity and mass are reduced in NPC1 fibroblasts. The GCase activity in normal and NPC1 fibroblasts (NPC1a and NPC1b) was determined in at least three separate flasks for each cell line. Data represent the means ⫾ S.D. Inset, representative Western blot of GCase from control and NPC1 fibroblast homogenates. Identical amounts of protein (5 ␮g) were loaded in each lane. Densitometric quantitation of the bands is also shown. Subcellular Localization of GCase, Sap C, and LBPA—Our previous findings showed that the activity of GCase is efficiently expressed only when the enzyme is bound to membranes containing anionic phospholipids (19, 32). Sap C, which preferentially interacts with these lipids (17), in turn promotes the association of GCase with the lipid surface. According to this model, it can be expected that GCase, Sap C, and LBPA, the most abundant anionic phospholipid of the endolysosomal compartment (33), colocalize in the same regions of the late endosomal/lysosomal membranes in control fibroblasts. As shown in Fig. 5, double-immunostaining revealed a complete colocalization of GCase and Sap C, as evident in the merged images. Moreover, all of the vesicular structures that contained GCase and Sap C also contained the anionic phospholipid LBPA. The late endosomal/lysosomal localization of GCase and Sap C was confirmed by the complete colocalization of the two proteins with LAMP1, a typical endolysosomal marker (Fig. 6). To investigate whether an altered subcellular distribution might be responsible of the GCase instability, we performed the same immunofluorescence tests in NPC1 cells. As shown in Fig. 7, extensive storage of free Chol was observed in the two NPC1 cell lines (NPC1a and NPC1b), as visualized by the characteristic staining with filipin. Fig. 7 also shows that some cells staining for Chol were almost devoid of GCase. This observation was quantified by scoring NPC1a and NPC1b cells for GCase staining (n ⫽ 20 fields for each cell line). The enzyme was nearly absent in about 50% of the cells, an observation in keeping with the low amount of GCase found in the fibroblast homogenates (see Fig. 1). Immunofluorescence microscopy furthermore revealed that GCase distributed toward the periphery of vesicles in enlarged rings containing a heavy burden of Chol. The segregation of GCase toward the periphery of vesicular structures was constantly observed in NPC1 fibroblasts. In cells in which a significant amount of GCase was present, double-immunostaining showed the non-coincidence of the GCase distribution with those of LBPA and Sap C (Fig. 8). Also, the colocalization of Sap C with LBPA was impaired (Fig. 8). The intracellular distribution of GCase and Sap C was further defined by Laser scanning confocal microscopy. As shown in Fig. 9, the contact among GCase and its activating and stabilizing factor, Sap C, is rare in NPC1 cells, whereas the two proteins completely colocalize in control fibroblasts. Because the depletion of Chol results in a dramatic increase of both the GCase mass and activity (see Fig. 2), we have investigated whether a reduction of the Chol level could also re-establish the colocalization of GCase with Sap C. The NPC1 cells were cultured for 7 days in medium containing LPDS. After this time, the morphology of the cells changed, and the filipin staining was no more detectable. As shown in Fig. 10, the decrease of Chol storage actually restored the colocalization of GCase with Sap C. Modulation of Glucosylceramidase in NPC Cells 17677 FIG. 2. Restoration of GCase activity and mass in NPC1 fibroblasts incubated in LPDS medium. The two NPC1 lines (NPC1a, ●, and NPC1b, f) were incubated in LPDS medium for the indicated periods of time (see “Experimental Procedures”). A, GCase activity was determined on lysates of cells harvested at the indicated days from time of subculture. B, GCase mass was determined on the same cell lysates by Western blotting. Identical amounts of protein (5 ␮g) were loaded in each lane. The samples were probed for GCase utilizing the monoclonal antibody 8E4. The blots were reprobed for ␤-actin to normalize lanes for protein content. The experiments, repeated more than three times, gave similar results. FIG. 3. Processing of GCase in control and NPC1 fibroblasts. Control, NPC1a, and NPC1b fibroblasts were pulsed for 1 h and then chased as indicated. Immunoprecipitation of cell lysates was performed with the anti-GCase monoclonal antibody 8E4. SDS-PAGE and fluorography were carried out as described under “Experimental Procedures.” The number on the left refers to the molecular mass (kDa) of albumin standard. The bands were quantitated by densitometry. The experiments, repeated more than two times, gave similar results. The Chol-mediated regulation of sphingomyelinase differs from that of GCase because the high amount of Chol required to knock down the sphingomyelinase activity has a negligible effect on the abundance and size of this enzyme (24). Actually, NPC fibroblasts express about 10% of the sphingomyelinase activity of normal fibroblasts but possess a normal amount of enzymatic protein. To explain their observations, the authors hypothesized that elevated free Chol might alter processing and/or trafficking events critical for sphingomyelinase activity or induce allosteric changes that cause enzyme inactivation (24). In normal cells, GCase is synthesized as a 62-kDa precursor and then converted into different molecular forms. In pulsechase experiments, the intensities of the GCase bands appearing after a 24-h chase increase with time, becoming stronger after chase periods of ⬎48 h (30). This phenomenon is possibly related to the accessibility and affinity of the different GCase forms for the anti-GCase antibody utilized. Our results show that in NPC1 fibroblasts, a normal amount of the precursor form of GCase is synthesized, whereas the abundance of the mature forms is low, indicating that the decreased GCase level is the consequence of an accelerated degradation of the mature enzyme. Moreover, we observed that the residual enzyme protein was not uniformly distributed among cells; some NPC1 fibroblasts contained significant amounts of GCase, whereas others were almost devoid of enzyme protein (see Fig. 7). When present, the enzyme was visualized at the periphery of Cholfilled vesicles. It has recently been reported that the intraendolysosomal membranes are organized as a mosaic of lipid domains with different lipid and protein composition (34). It can be envisaged that the accumulation of Chol, altering the organization of the lipid domains, either brings about a redistribution of GCase toward the more external membranes of the endolysosomal vesicles or increases the susceptibility of GCase present in the core of the vesicles to the protease attack. Interestingly, it has also been found that mutant inactive forms of the NPC1 protein, transiently expressed in CT60 cells (a Chinese hamster ovary cell mutant), are localized in endolysosomal membranes encircling Chol-laden cores (35, 36). The fast disappearance of GCase in NPC1 fibroblasts suggests that the enzyme is localized in a less protective environment in these cells. It is known that GCase is stabilized when in contact with anionic phospholipids and its activator protein, Downloaded from http://www.jbc.org/ by guest on June 7, 2020 FIG. 4. Processing of prosaposin in control and NPC1 fibroblasts. Control, NPC1a, and NPC1b fibroblasts were pulsed for 1 h and then chased as indicated. Immunoprecipitation of cell lysates was performed with anti-Sap C antibody. SDS-PAGE and fluorography were carried out as described under “Experimental Procedures.” The numbers on the left refer to the molecular mass (kDa) of standards. The percentage of prosaposin (molecular mass ⫽ 73– 65 kDa) conversion to Sap C (molecular mass ⫽ 14 –10 kDa) was evaluated by the intensities of the corresponding bands quantitated by densitometry. The experiments, repeated more than two times, gave similar results. 17678 Modulation of Glucosylceramidase in NPC Cells FIG. 6. GCase and Sap C colocalize with LAMP 1 in normal fibroblasts. Normal human fibroblasts were double-immunostained for LAMP1 and either Sap C (top panels) or GCase (bottom panels), as described under “Experimental Procedures.” All vesicles containing LAMP1 were also GCase-positive and Sap C-positive. Note that GCase was visualized with a polyclonal antibody (red, bottom left panel). Bars, 10 ␮m. Sap C (37). Moreover, a recent work indicates that Sap C is required for GCase resistance to proteolytic degradation in the cells (38). The instability of GCase in NPC1 fibroblasts cannot be attributed to a low amount of Sap C, because we found that high levels of prosaposin are synthesized and converted to Sap C in these cells (see Fig. 4), the only difference from control fibroblasts being a delay in Sap C maturation. The sloweddown processing of prosaposin suggests that the normal transport of the protein from the endoplasmic reticulum to the late endosomes/lysosomes and/or its proteolysis in these organelles are retarded. To exert its activating and anti-proteolytic protective function, Sap C should be in contact with GCase. Actually, fluorescence microscopy has now shown a complete colocalization of GCase with Sap C in normal fibroblasts. Conversely, in NPC1 fibroblasts, most of the GCase-containing structures were not Sap C-positive, indicating that the two proteins preferentially FIG. 8. GCase, Sap C, and LBPA poorly colocalize in NPC1 fibroblasts. NPC1 fibroblasts were double-immunostained for GCase and Sap C (top panels), GCase and LBPA (middle panels), or Sap C and LBPA (bottom panels), as described under “Experimental Procedures.” The right panels show an enlargement of the regions outlined by the boxes in the overlaid panels to better appreciate the poor colocalization of GCase, Sap C, and LBPA. GCase was visualized with a monoclonal antibody (green, left, top panel) or with a polyclonal antibody (red, left, middle panel). As already observed in Fig. 7, GCase distributes toward the periphery of the vesicles. Bars, 10 ␮m. distribute into distinct vesicular compartments. The occurrence of separate subsets of endolysosomal vesicles with partly different protein and lipid content is well documented (4, 39). Most likely, the localization of GCase on membranes devoid of Sap C decreases the enzyme stability. As we have previously shown, the localization of GCase on membranes is regulated by several factors, the more important being pH, anionic phospholipids, and Sap C. In fact, low pH values similar to those of the endolysosomal compartment dramatically increase Sap C hydrophobicity (17, 40). In consequence, the saposin associates with and destabilizes anionic phospholipid-containing membranes, promoting in turn the association of GCase with the lipid surface (19, 32). The amount and the physical organization of anionic phospholipids have a key role in the Sap C-mediated binding of GCase to give rise to the enzymatically active complex. Our present results indicate that GCase does not colocalize with either Sap C or LBPA, the main anionic phospholipid of the endolysosomal organelles (33), in NPC1 cells. Most likely, the accumulation of Chol Downloaded from http://www.jbc.org/ by guest on June 7, 2020 FIG. 5. GCase, Sap C, and LBPA colocalize in the same vesicular structures in normal fibroblasts. Normal human fibroblasts were double-immunostained for GCase and Sap C (top panels), GCase and LBPA (middle panels), and Sap C and LBPA (bottom panels), as described under “Experimental Procedures.” All vesicles containing GCase were also Sap C- and LBPA-positive. The right panels show an enlargement of the region outlined by the boxes in the overlaid panels to better appreciate the complete colocalization of GCase, Sap C, and LBPA. Note that GCase was visualized with the monoclonal antibody 8E4 (green, top left panel) or with a polyclonal antibody (red, middle left panel). Bars, 10 ␮m. FIG. 7. GCase and Chol in NPC1 fibroblasts. NPC1 fibroblasts (NPC1a, top panels; NPC1b, bottom panels) were immunostained for GCase and cytochemically stained with filipin for Chol as described under “Experimental Procedures.” Arrowheads highlight cells containing vesicular structures filled with Chol but almost devoid of GCase. The right panels show an enlargement of the regions outlined by the boxes in the overlaid panels. As better revealed in the magnified images, GCase appears as rings at the periphery of Chol-laden vesicular structures. Bars, 10 ␮m. Modulation of Glucosylceramidase in NPC Cells FIG. 9. Comparison of the GCase and Sap C localization in normal and NPC1 fibroblasts. NPC1 (top panels) and normal (bottom panels) fibroblasts were double-immunostained for GCase (green) and Sap C (red) and observed by laser scanning microscopy as described under “Experimental Procedures.” The right panels show an enlargement of the regions outlined by the boxes in the overlaid panels. The comparison of the overlaid images clearly shows that, in normal fibroblasts, the intracellular vesicles are yellow, indicating that each contains both GCase and Sap C, whereas in NPC1 cells, most of the vesicles are either green or red, indicating that the two proteins reside in distinct vesicles. Bars, 10 ␮m. FIG. 10. Chol depletion from NPC fibroblasts restores the colocalization between GCase and Sap C. NPC1 fibroblasts were grown for 7 days in medium either supplemented with FBS (left panel) or with LPDS (right panel). The cells were then double-immunostained for GCase (green) and Sap C (red). The comparison of the overlaid images clearly shows that the incubation with LPDS dramatically increases the number of intracellular yellow vesicles, namely of vesicles that contain both Sap C and GCase. Bars, 10 ␮m. activity by controlling the organization of the endolysosomal membranes. In conclusion, our findings indicate, for the first time, that in NPC1 fibroblasts, the amount of GCase protein is markedly decreased, and the colocalization of GCase with LBPA and Sap C is impaired as consequence of Chol accumulation. These findings strongly suggest that the disruption of the complex formed by the enzyme and its stabilizing and activating factors (anionic phospholipids and Sap C) might be the cause of the decreased GCase activity observed in NPC fibroblasts and might participate in the GC accumulation observed in NPC tissues. Acknowledgment—We thank V. Raia for technical assistance. REFERENCES 1. Patterson, M. C., Vanier, M. T., Suzuki, K., Morris, J. A., Carstea, E., Neufeld, E. 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Chem. 2004, 279:17674-17680. doi: 10.1074/jbc.M313517200 originally published online February 2, 2004 Access the most updated version of this article at doi: 10.1074/jbc.M313517200 Alerts: • When this article is cited • When a correction for this article is posted This article cites 42 references, 15 of which can be accessed free at http://www.jbc.org/content/279/17/17674.full.html#ref-list-1 Downloaded from http://www.jbc.org/ by guest on June 7, 2020 Click here to choose from all of JBC's e-mail alerts